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1
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Pepler MAD, Mulholland EL, Montague FR, Elliot MA. Defining the networks that connect RNase III and RNase J-mediated regulation of primary and specialized metabolism in Streptomyces venezuelae. J Bacteriol 2025; 207:e0002425. [PMID: 40227046 PMCID: PMC12096830 DOI: 10.1128/jb.00024-25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2025] [Accepted: 03/18/2025] [Indexed: 04/15/2025] Open
Abstract
RNA metabolism involves coordinating RNA synthesis with RNA processing and degradation. Ribonucleases play fundamental roles within the cell, contributing to the cleavage, modification, and degradation of RNA molecules, with these actions ensuring appropriate gene regulation and cellular homeostasis. Here, we employed RNA sequencing to explore the impact of RNase III and RNase J on the transcriptome of Streptomyces venezuelae. Differential expression analysis comparing wild-type and RNase mutant strains at distinct developmental stages revealed significant changes in transcript abundance, particularly in pathways related to multicellular development, nutrient acquisition, and specialized metabolism. Both RNase mutants exhibited dysregulation of the BldD regulon, including altered expression of many cyclic-di-GMP-associated enzymes. We also observed precocious chloramphenicol production in these RNase mutants and found that in the RNase III mutant, this was associated with PhoP-mediated regulation. We further found that RNase III directly targeted members of the PhoP regulon, suggesting a link between RNA metabolism and a regulator that bridges primary and specialized metabolism. We connected RNase J function with translation through the observation that RNase J directly targets multiple ribosomal protein transcripts for degradation. These findings establish distinct but complementary roles for RNase III and RNase J in coordinating the gene expression dynamics critical for S. venezuelae development and specialized metabolism. IMPORTANCE RNA processing and metabolism are mediated by ribonucleases and are fundamental processes in all cells. In the morphologically complex and metabolically sophisticated Streptomyces bacteria, RNase III and RNase J influence both development and metabolism through poorly understood mechanisms. Here, we show that both ribonucleases are required for the proper expression of the BldD developmental pathway and contribute to the control of chloramphenicol production, with an interesting connection to phosphate regulation for RNase III. Additionally, we show that both RNases have the potential to impact translation through distinct mechanisms and can function cooperatively in degrading specific transcripts. This study advances our understanding of RNases in Streptomyces biology by providing insight into distinct contributions made by these enzymes and the intriguing interplay between them.
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Affiliation(s)
- Meghan A. D. Pepler
- Department of Biology, McMaster University, Hamilton, Ontario, Canada
- Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada
| | - Emma L. Mulholland
- Department of Biology, McMaster University, Hamilton, Ontario, Canada
- Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada
| | - Freddie R. Montague
- Department of Biology, McMaster University, Hamilton, Ontario, Canada
- Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada
| | - Marie A. Elliot
- Department of Biology, McMaster University, Hamilton, Ontario, Canada
- Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada
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2
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D’Agostino F, Pinatel E, Meynhardt A, Scarlato V, Vannini A, Roncarati D. RNase Y mediates posttranscriptional control of the virulence-associated CncR1 small-RNA in Helicobacter pylori. iScience 2025; 28:111815. [PMID: 39949958 PMCID: PMC11821409 DOI: 10.1016/j.isci.2025.111815] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2024] [Revised: 11/15/2024] [Accepted: 01/10/2025] [Indexed: 02/16/2025] Open
Abstract
Ribonucleases are involved in several biological processes, including the turnover of structural and messenger RNAs and the specific processing of the cellular transcriptome. Here, we characterized the RNase Y from Helicobacter pylori. We found that RNase Y is membrane-associated and its expression is controlled during bacterial growth and by Fur in response to iron. We observed that RNase Y deletion has a limited impact on H. pylori transcriptome and on bacterial growth. Interestingly, we found that RNase Y is involved in the metabolism of CncR1, a virulence-associated sRNA oppositely modulating bacterial motility and adhesion to host cells. Indeed, RNase Y inactivation led to the accumulation of a 3'-extended CncR1 isoform, which appeared unable to interact in vitro with a known target mRNA. The observation that the RNAse Y-mutant strain showed deregulation of several members of the CncR1 regulon suggests this ribonuclease has an important role in H. pylori posttranscriptional regulation.
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Affiliation(s)
- Federico D’Agostino
- Department of Pharmacy and Biotechnology, Alma Mater Studiorum – University of Bologna, Bologna, Italy
| | - Eva Pinatel
- Institute of Biomedical Technologies - National Research Council, Segrate, Italy
| | - Alexandra Meynhardt
- Department of Pharmacy and Biotechnology, Alma Mater Studiorum – University of Bologna, Bologna, Italy
| | - Vincenzo Scarlato
- Department of Pharmacy and Biotechnology, Alma Mater Studiorum – University of Bologna, Bologna, Italy
| | - Andrea Vannini
- Department of Pharmacy and Biotechnology, Alma Mater Studiorum – University of Bologna, Bologna, Italy
| | - Davide Roncarati
- Department of Pharmacy and Biotechnology, Alma Mater Studiorum – University of Bologna, Bologna, Italy
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3
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Taggart J, Dierksheide K, LeBlanc H, Lalanne JB, Durand S, Braun F, Condon C, Li GW. A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis. Nucleic Acids Res 2025; 53:gkaf030. [PMID: 39883015 PMCID: PMC11780869 DOI: 10.1093/nar/gkaf030] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2024] [Revised: 01/03/2025] [Accepted: 01/17/2025] [Indexed: 01/31/2025] Open
Abstract
RNA endonucleases are the rate-limiting initiator of decay for many bacterial mRNAs. However, the positions of cleavage and their sequence determinants remain elusive even for the well-studied Bacillus subtilis. Here we present two complementary approaches-transcriptome-wide mapping of endoribonucleolytic activity and deep mutational scanning of RNA cleavage sites-that reveal distinct rules governing the specificity among B. subtilis endoribonucleases. Detection of RNA terminal nucleotides in both 5'- and 3'-exonuclease-deficient cells revealed >103 putative endonucleolytic cleavage sites with single-nucleotide resolution. We found a surprisingly weak consensus for RNase Y targets, a contrastingly strong primary sequence motif for EndoA targets, and long-range intramolecular secondary structures for RNase III targets. Deep mutational analysis of RNase Y cleavage sites showed that the specificity is governed by many disjointed sequence features. Our results highlight the delocalized nature of mRNA stability determinants and provide a strategy for elucidating endoribonuclease specificity in vivo.
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Affiliation(s)
- James C Taggart
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
| | | | - Hannah J LeBlanc
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
| | - Jean-Benoît Lalanne
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
- Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
| | - Sylvain Durand
- Expression Génétique Microbienne (EGM), CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
| | - Frédérique Braun
- Expression Génétique Microbienne (EGM), CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
| | - Ciarán Condon
- Expression Génétique Microbienne (EGM), CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
| | - Gene-Wei Li
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
- Howard Hughes Medical Institute, Cambridge, MA 02142, USA
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4
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D’Halluin A, Gilet L, Lablaine A, Pellegrini O, Serrano M, Tolcan A, Ventroux M, Durand S, Hamon M, Henriques A, Carballido-López R, Condon C. Embedding a ribonuclease in the spore crust couples gene expression to spore development in Bacillus subtilis. Nucleic Acids Res 2025; 53:gkae1301. [PMID: 39817517 PMCID: PMC11736430 DOI: 10.1093/nar/gkae1301] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2024] [Revised: 12/16/2024] [Accepted: 01/14/2025] [Indexed: 01/18/2025] Open
Abstract
Faced with nutritional stress, some bacteria form endospores capable of enduring extreme conditions for long periods of time; yet the function of many proteins expressed during sporulation remains a mystery. We identify one such protein, KapD, as a 3'-exoribonuclease expressed under control of the mother cell-specific transcription factors SigE and SigK in Bacillus subtilis. KapD dynamically assembles over the spore surface through a direct interaction with the major crust protein CotY. KapD catalytic activity is essential for normal adhesiveness of spore surface layers. We identify the sigK mRNA as a key KapD substrate and and show that the stability of this transcript is regulated by CotY-mediated sequestration of KapD. SigK is tightly controlled through excision of a prophage-like element, transcriptional regulation and the removal of an inhibitory pro-sequence. Our findings uncover a fourth, post-transcriptional layer of control of sigK expression that couples late-stage gene expression in the mother cell to spore morphogenesis.
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Affiliation(s)
- Alexandre D’Halluin
- EGM CNRS, Université Paris-Cité,Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
| | - Laetitia Gilet
- EGM CNRS, Université Paris-Cité,Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
| | - Armand Lablaine
- Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France
| | - Olivier Pellegrini
- EGM CNRS, Université Paris-Cité,Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
| | - Mónica Serrano
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157Oeiras, Portugal
| | - Anastasia Tolcan
- EGM CNRS, Université Paris-Cité,Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
| | - Magali Ventroux
- Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France
| | - Sylvain Durand
- EGM CNRS, Université Paris-Cité,Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
| | - Marion Hamon
- Proteomics platform, FR550 Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
| | - Adriano O Henriques
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157Oeiras, Portugal
| | - Rut Carballido-López
- Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France
| | - Ciarán Condon
- EGM CNRS, Université Paris-Cité,Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
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5
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Liu YJ, Wang X, Sun Y, Feng Y. Bacterial 5' UTR: A treasure-trove for post-transcriptional regulation. Biotechnol Adv 2025; 78:108478. [PMID: 39551455 DOI: 10.1016/j.biotechadv.2024.108478] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2024] [Revised: 11/08/2024] [Accepted: 11/12/2024] [Indexed: 11/19/2024]
Abstract
In bacteria, where gene transcription and translation occur concurrently, post-transcriptional regulation is acknowledged to be effective and precise. The 5' untranslated regions (5' UTRs) typically harbor diverse post-transcriptional regulatory elements, like riboswitches, RNA thermometers, small RNAs, and upstream open reading frames, that serve to modulate transcription termination, translation initiation, and mRNA stability. Consequently, exploring 5' UTR-derived regulatory elements is vital for synthetic biology and metabolic engineering. Over the past few years, the investigation of successive mechanisms has facilitated the development of various genetic tools from bacterial 5' UTRs. This review consolidates current understanding of 5' UTR regulatory functions, presents recent progress in 5' UTR-element design and screening, updates the tools and regulatory strategies developed, and highlights the challenges and necessity of establishing reliable bioinformatic analysis methods and non-model bacterial chassis in the future.
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Affiliation(s)
- Ya-Jun Liu
- CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China; Shandong Energy Institute, Qingdao 266101, China; Qingdao New Energy Shandong Laboratory, Qingdao 266101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
| | - Xiaoqing Wang
- CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China; Shandong Energy Institute, Qingdao 266101, China; Qingdao New Energy Shandong Laboratory, Qingdao 266101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yuman Sun
- CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China; Shandong Energy Institute, Qingdao 266101, China; Qingdao New Energy Shandong Laboratory, Qingdao 266101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yingang Feng
- CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China; Shandong Energy Institute, Qingdao 266101, China; Qingdao New Energy Shandong Laboratory, Qingdao 266101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
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6
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Hasan MK, Jeannine Brady L. Nucleic acid-binding KH domain proteins influence a spectrum of biological pathways including as part of membrane-localized complexes. J Struct Biol X 2024; 10:100106. [PMID: 39040530 PMCID: PMC11261784 DOI: 10.1016/j.yjsbx.2024.100106] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2024] [Revised: 06/18/2024] [Accepted: 06/24/2024] [Indexed: 07/24/2024] Open
Abstract
K-Homology domain (KH domain) proteins bind single-stranded nucleic acids, influence protein-protein interactions of proteins that harbor them, and are found in all kingdoms of life. In concert with other functional protein domains KH domains contribute to a variety of critical biological activities, often within higher order machineries including membrane-localized protein complexes. Eukaryotic KH domain proteins are linked to developmental processes, morphogenesis, and growth regulation, and their aberrant expression is often associated with cancer. Prokaryotic KH domain proteins are involved in integral cellular activities including cell division and protein translocation. Eukaryotic and prokaryotic KH domains share structural features, but are differentiated based on their structural organizations. In this review, we explore the structure/function relationships of known examples of KH domain proteins, and highlight cases in which they function within or at membrane surfaces. We also summarize examples of KH domain proteins that influence bacterial virulence and pathogenesis. We conclude the article by discussing prospective research avenues that could be pursued to better investigate this largely understudied protein category.
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Affiliation(s)
- Md Kamrul Hasan
- Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA
- Division of Biology, Kansas State University, Manhattan, KS 66506, USA
| | - L. Jeannine Brady
- Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA
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7
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Laalami S, Cavaiuolo M, Oberto J, Putzer H. Membrane Localization of RNase Y Is Important for Global Gene Expression in Bacillus subtilis. Int J Mol Sci 2024; 25:8537. [PMID: 39126106 PMCID: PMC11313650 DOI: 10.3390/ijms25158537] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2024] [Revised: 07/27/2024] [Accepted: 07/29/2024] [Indexed: 08/12/2024] Open
Abstract
RNase Y is a key endoribonuclease that regulates global mRNA turnover and processing in Bacillus subtilis and likely many other bacteria. This enzyme is anchored to the cell membrane, creating a pseudo-compartmentalization that aligns with its role in initiating the decay of mRNAs primarily translated at the cell periphery. However, the reasons behind and the consequences of RNase Y's membrane attachment remain largely unknown. In our study, we examined a strain expressing wild-type levels of a cytoplasmic form of RNase Y from its chromosomal locus. This strain exhibits a slow-growth phenotype, similar to that of an RNase Y null mutant. Genome-wide data reveal a significant impact on the expression of hundreds of genes. While certain RNA substrates clearly depend on RNase Y's membrane attachment, others do not. We observed no correlation between mRNA stabilization in the mutant strains and the cellular location or function of the encoded proteins. Interestingly, the Y-complex, a specificity factor for RNase Y, also appears also recognize the cytoplasmic form of the enzyme, restoring wild-type levels of the corresponding transcripts. We propose that membrane attachment of RNase Y is crucial for its functional interaction with many coding and non-coding RNAs, limiting the cleavage of specific substrates, and potentially avoiding unfavorable competition with other ribonucleases like RNase J, which shares a similar evolutionarily conserved cleavage specificity.
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Affiliation(s)
- Soumaya Laalami
- Expression Génétique Microbienne, CNRS, Institut de Biologie Physico-Chimique, Université Paris Cité, 75005 Paris, France; (S.L.)
| | - Marina Cavaiuolo
- Expression Génétique Microbienne, CNRS, Institut de Biologie Physico-Chimique, Université Paris Cité, 75005 Paris, France; (S.L.)
- Laboratory for Food Safety, SBCL Unit, University Paris Est, ANSES, 94701 Maisons-Alfort, France
| | - Jacques Oberto
- Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, 91198 Gif-sur-Yvette, France;
| | - Harald Putzer
- Expression Génétique Microbienne, CNRS, Institut de Biologie Physico-Chimique, Université Paris Cité, 75005 Paris, France; (S.L.)
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8
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Le Scornet A, Jousselin A, Baumas K, Kostova G, Durand S, Poljak L, Barriot R, Coutant E, Pigearias R, Tejero G, Lootvoet J, Péllisier C, Munoz G, Condon C, Redder P. Critical factors for precise and efficient RNA cleavage by RNase Y in Staphylococcus aureus. PLoS Genet 2024; 20:e1011349. [PMID: 39088561 PMCID: PMC11321564 DOI: 10.1371/journal.pgen.1011349] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2024] [Revised: 08/13/2024] [Accepted: 06/23/2024] [Indexed: 08/03/2024] Open
Abstract
Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y.
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Affiliation(s)
- Alexandre Le Scornet
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Ambre Jousselin
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Kamila Baumas
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Gergana Kostova
- UMR8261 CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, France
| | - Sylvain Durand
- UMR8261 CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, France
| | - Leonora Poljak
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Roland Barriot
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Eve Coutant
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Romain Pigearias
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Gabriel Tejero
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Jonas Lootvoet
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Céline Péllisier
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Gladys Munoz
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
| | - Ciarán Condon
- UMR8261 CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, France
| | - Peter Redder
- Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, Université Toulouse III–Paul Sabatier, Toulouse, France
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9
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Quarta G, Schlick T. Riboswitch Distribution in the Human Gut Microbiome Reveals Common Metabolite Pathways. J Phys Chem B 2024; 128:4336-4343. [PMID: 38657162 PMCID: PMC11089507 DOI: 10.1021/acs.jpcb.4c00267] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2024] [Revised: 03/16/2024] [Accepted: 03/21/2024] [Indexed: 04/26/2024]
Abstract
Riboswitches are widely distributed, conserved RNAs which regulate metabolite levels in bacterial cells through direct, noncovalent binding of their cognate metabolite. Various riboswitch families are highly enriched in gut bacteria, suggestive of a symbiotic relationship between the host and bacteria. Previous studies of the distribution of riboswitches have examined bacterial taxa broadly. Thus, the distribution of riboswitches associated with bacteria inhabiting the intestines of healthy individuals is not well understood. To address these questions, we survey the gut microbiome for riboswitches by including an international database of prokaryotic genomes from the gut samples. Using Infernal, a program that uses RNA-specific sequence and structural features, we survey this data set using existing riboswitch models. We identify 22 classes of riboswitches with vitamin cofactors making up the majority of riboswitch-associated pathways. Our finding is reproducible in other representative databases from the oral as well as the marine microbiomes, underscoring the importance of thiamine pyrophosphate, cobalamin, and flavin mononucleotide in gene regulation. Interestingly, riboswitches do not vary significantly across microbiome representatives from around the world despite major taxonomic differences; this suggests an underlying conservation. Further studies elucidating the role of bacterial riboswitches in the host metabolome are needed to illuminate the consequences of our finding.
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Affiliation(s)
- Giulio Quarta
- Department
of Medicine, NYU Grossman School of Medicine, 450 East 29th St., Room 341, New York, New York 10016, United States
| | - Tamar Schlick
- Department
of Chemistry, New York University, 100 Washington Square East, Silver
Building, New York, New York 10003, United States
- Courant
Institute of Mathematical Sciences, New
York University, 251
Mercer Street, New York, New York 10012, United States
- New
York University-East China Normal University Center for Computational
Chemistry, New York University Shanghai, Shanghai 200122, China
- Simons
Center for Computational Physical Chemistry, New York University, 24 Waverly Place, Silver Building, New York, New York 10003, United States
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10
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Vaňková Hausnerová V, Shoman M, Kumar D, Schwarz M, Modrák M, Jirát Matějčková J, Mikesková E, Neva S, Herrmannová A, Šiková M, Halada P, Novotná I, Pajer P, Valášek LS, Převorovský M, Krásný L, Hnilicová J. RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria. Nucleic Acids Res 2024; 52:4604-4626. [PMID: 38348908 PMCID: PMC11077062 DOI: 10.1093/nar/gkae081] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2023] [Revised: 01/24/2024] [Accepted: 01/29/2024] [Indexed: 05/09/2024] Open
Abstract
Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.
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Affiliation(s)
- Viola Vaňková Hausnerová
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
- Laboratory of Regulatory RNAs, Faculty of Science, Charles University, Prague128 44, Czech Republic
| | - Mahmoud Shoman
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
- Laboratory of Regulatory RNAs, Faculty of Science, Charles University, Prague128 44, Czech Republic
| | - Dilip Kumar
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
| | - Marek Schwarz
- Laboratory of Bioinformatics, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
| | - Martin Modrák
- Laboratory of Bioinformatics, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
- Department of Bioinformatics, Second Faculty of Medicine, Charles University, Prague150 06, Czech Republic
| | - Jitka Jirát Matějčková
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
- Laboratory of Regulatory RNAs, Faculty of Science, Charles University, Prague128 44, Czech Republic
| | - Eliška Mikesková
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
- Laboratory of Regulatory RNAs, Faculty of Science, Charles University, Prague128 44, Czech Republic
| | - Silvia Neva
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
- Laboratory of Regulatory RNAs, Faculty of Science, Charles University, Prague128 44, Czech Republic
| | - Anna Herrmannová
- Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
| | - Michaela Šiková
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
| | - Petr Halada
- Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology of the Czech Academy of Sciences, Vestec252 50, Czech Republic
| | - Iva Novotná
- Military Health Institute, Military Medical Agency, Prague169 02, Czech Republic
| | - Petr Pajer
- Military Health Institute, Military Medical Agency, Prague169 02, Czech Republic
| | - Leoš Shivaya Valášek
- Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
| | - Martin Převorovský
- Department of Cell Biology, Faculty of Science, Charles University, Prague128 00, Czech Republic
| | - Libor Krásný
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
| | - Jarmila Hnilicová
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague142 20, Czech Republic
- Laboratory of Regulatory RNAs, Faculty of Science, Charles University, Prague128 44, Czech Republic
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11
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Walgraeve J, Ferrero-Bordera B, Maaß S, Becher D, Schwerdtfeger R, van Dijl JM, Seefried M. Diamide-based screening method for the isolation of improved oxidative stress tolerance phenotypes in Bacillus mutant libraries. Microbiol Spectr 2023; 11:e0160823. [PMID: 37819171 PMCID: PMC10714788 DOI: 10.1128/spectrum.01608-23] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Accepted: 08/30/2023] [Indexed: 10/13/2023] Open
Abstract
IMPORTANCE During their life cycle, bacteria are exposed to a range of different stresses that need to be managed appropriately in order to ensure their growth and viability. This applies not only to bacteria in their natural habitats but also to bacteria employed in biotechnological production processes. Oxidative stress is one of these stresses that may originate either from bacterial metabolism or external factors. In biotechnological settings, it is of critical importance that production strains are resistant to oxidative stresses. Accordingly, this also applies to the major industrial cell factory Bacillus subtilis. In the present study, we, therefore, developed a screen for B. subtilis strains with enhanced oxidative stress tolerance. The results show that our approach is feasible and time-, space-, and resource-efficient. We, therefore, anticipate that it will enhance the development of more robust industrial production strains with improved robustness under conditions of oxidative stress.
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Affiliation(s)
| | | | - Sandra Maaß
- Department of Microbial Proteomics, University of Greifswald, Greifswald, Germany
| | - Dörte Becher
- Department of Microbial Proteomics, University of Greifswald, Greifswald, Germany
| | | | - Jan Maarten van Dijl
- Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
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12
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Wiegard JC, Damm K, Lechner M, Thölken C, Ngo S, Putzer H, Hartmann RK. Processing and decay of 6S-1 and 6S-2 RNAs in Bacillus subtilis. RNA (NEW YORK, N.Y.) 2023; 29:1481-1499. [PMID: 37369528 PMCID: PMC10578484 DOI: 10.1261/rna.079666.123] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/22/2023] [Accepted: 06/06/2023] [Indexed: 06/29/2023]
Abstract
Noncoding 6S RNAs regulate transcription by binding to the active site of bacterial RNA polymerase holoenzymes. Processing and decay of 6S-1 and 6S-2 RNA were investigated in Bacillus subtilis by northern blot and RNA-seq analyses using different RNase knockout strains, as well as by in vitro processing assays. For both 6S RNA paralogs, we identified a key-but mechanistically different-role of RNase J1. RNase J1 catalyzes 5'-end maturation of 6S-1 RNA, yet relatively inefficient and possibly via the enzyme's "sliding endonuclease" activity. 5'-end maturation has no detectable effect on 6S-1 RNA function, but rather regulates its decay: The generated 5'-monophosphate on matured 6S-1 RNA propels endonucleolytic cleavage in its apical loop region. The major 6S-2 RNA degradation pathway is initiated by endonucleolytic cleavage in the 5'-central bubble to trigger 5'-to-3'-exoribonucleolytic degradation of the downstream fragment by RNase J1. The four 3'-exonucleases of B. subtilis-RNase R, PNPase, YhaM, and particularly RNase PH-are involved in 3'-end trimming of both 6S RNAs, degradation of 6S-1 RNA fragments, and decay of abortive transcripts (so-called product RNAs, ∼14 nt in length) synthesized on 6S-1 RNA during outgrowth from stationary phase. In the case of the growth-retarded RNase Y deletion strain, we were unable to infer a specific role of RNase Y in 6S RNA decay. Yet, a participation of RNase Y in 6S RNA decay still remains possible, as evidence for such a function may have been obscured by overlapping substrate specificities of RNase Y, RNase J1, and RNase J2.
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Affiliation(s)
- Jana Christin Wiegard
- Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, D-35037 Marburg, Germany
| | - Katrin Damm
- Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, D-35037 Marburg, Germany
| | - Marcus Lechner
- Philipps-Universität Marburg, Center for Synthetic Microbiology (SYNMIKRO), Bioinformatics Core Facility, D-35032 Marburg, Germany
| | - Clemens Thölken
- Philipps-Universität Marburg, Center for Synthetic Microbiology (SYNMIKRO), Bioinformatics Core Facility, D-35032 Marburg, Germany
| | - Saravuth Ngo
- Expression Génétique Microbienne, CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, 75005 Paris, France
| | - Harald Putzer
- Expression Génétique Microbienne, CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, 75005 Paris, France
| | - Roland K Hartmann
- Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, D-35037 Marburg, Germany
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13
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van Gestel J, Wagner A, Ackermann M. Pleiotropic hubs drive bacterial surface competition through parallel changes in colony composition and expansion. PLoS Biol 2023; 21:e3002338. [PMID: 37844064 PMCID: PMC10578586 DOI: 10.1371/journal.pbio.3002338] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2023] [Accepted: 09/18/2023] [Indexed: 10/18/2023] Open
Abstract
Bacteria commonly adhere to surfaces where they compete for both space and resources. Despite the importance of surface growth, it remains largely elusive how bacteria evolve on surfaces. We previously performed an evolution experiment where we evolved distinct Bacilli populations under a selective regime that favored colony spreading. In just a few weeks, colonies of Bacillus subtilis showed strongly advanced expansion rates, increasing their radius 2.5-fold relative to that of the ancestor. Here, we investigate what drives their rapid evolution by performing a uniquely detailed analysis of the evolutionary changes in colony development. We find mutations in diverse global regulators, RicT, RNAse Y, and LexA, with strikingly similar pleiotropic effects: They lower the rate of sporulation and simultaneously facilitate colony expansion by either reducing extracellular polysaccharide production or by promoting filamentous growth. Combining both high-throughput flow cytometry and gene expression profiling, we show that regulatory mutations lead to highly reproducible and parallel changes in global gene expression, affecting approximately 45% of all genes. This parallelism results from the coordinated manner by which regulators change activity both during colony development-in the transition from vegetative growth to dormancy-and over evolutionary time. This coordinated activity can however also break down, leading to evolutionary divergence. Altogether, we show how global regulators function as major pleiotropic hubs that drive rapid surface adaptation by mediating parallel changes in both colony composition and expansion, thereby massively reshaping gene expression.
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Affiliation(s)
- Jordi van Gestel
- Department of Evolutionary Biology and Environmental Studies, University of Zürich, Zürich, Switzerland
- Swiss Institute of Bioinformatics, Lausanne, Switzerland
- Department of Environmental Systems Science, ETH Zürich, Zürich, Switzerland
- Department of Environmental Microbiology, Swiss Federal Institute of Aquatic Science and Technology (Eawag), Dübendorf, Switzerland
- Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America
- Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Andreas Wagner
- Department of Evolutionary Biology and Environmental Studies, University of Zürich, Zürich, Switzerland
- Swiss Institute of Bioinformatics, Lausanne, Switzerland
- The Santa Fe Institute, Santa Fe, New Mexico, United States of America
- Stellenbosch Institute for Advanced Study (STIAS), Wallenberg Research Centre at Stellenbosch University, Stellenbosch, South Africa
| | - Martin Ackermann
- Department of Environmental Systems Science, ETH Zürich, Zürich, Switzerland
- Department of Environmental Microbiology, Swiss Federal Institute of Aquatic Science and Technology (Eawag), Dübendorf, Switzerland
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14
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Brandwein JN, Sculthorpe TS, Ridder MJ, Bose JL, Rice KC. Factors impacting the regulation of nos gene expression in Staphylococcus aureus. Microbiol Spectr 2023; 11:e0168823. [PMID: 37747881 PMCID: PMC10580903 DOI: 10.1128/spectrum.01688-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2023] [Accepted: 07/29/2023] [Indexed: 09/27/2023] Open
Abstract
Staphylococcus aureus nitric oxide synthase (saNOS) contributes to oxidative stress resistance, antibiotic tolerance, virulence, and modulation of aerobic and nitrate-based cellular respiration. Despite its involvement in these essential processes, the genetic regulation of nos expression has not been well characterized. 5' rapid amplification of cDNA ends on nos RNA isolated from S. aureus UAMS-1 (USA200 strain) and AH1263 (USA300 strain) revealed that the nos transcriptional start site mapped to an adenine nucleotide in the predicted Shine-Dalgarno site located 11 bp upstream of the nos ATG start codon, suggesting that the nos transcript may have a leaderless organization or may be subject to processing. The SrrAB two-component system (TCS) was previously identified as a positive regulator of nos RNA levels, and experiments using a β-galactosidase reporter plasmid confirmed that SrrAB is a positive regulator of nos promoter activity. In addition, the quorum-sensing system Agr was identified as a negative regulator of low-oxygen nos expression in UAMS-1, with activity epistatic to SrrAB. Involvement of Agr was strain dependent, as nos expression remained unchanged in an AH1263 agr mutant, which has higher Agr activity compared to UAMS-1. Furthermore, nos promoter activity and RNA levels were significantly stronger in AH1263 relative to UAMS-1 during late-exponential low-oxygen growth, when nos expression is maximal. Global regulators Rex and MgrA were also implicated as negative regulators of low-oxygen nos promoter activity in UAMS-1. Collectively, these results provide new insight into factors that control nos expression.IMPORTANCEBacterial nitric oxide synthase (bNOS) has recently emerged in several species as a key player in resistance to stresses commonly encountered during infection. Although Staphylococcus aureus (sa)NOS has been suggested to be a promising drug target in S. aureus, an obstacle to this in practice is the existence of mammalian NOS, whose oxygenase domain is like bacterial NOS. Increased understanding of the nos regulatory network in S. aureus could allow targeting of saNOS through its regulators, bypassing the issue of also inhibiting mammalian NOS. Furthermore, the observed strain-dependent differences in S. aureus nos regulation presented in this study reinforce the importance of studying bacterial NOS regulation and function at both the strain and species levels.
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Affiliation(s)
- Jessica N. Brandwein
- Department of Microbiology & Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA
| | - Tiffany S. Sculthorpe
- Department of Microbiology & Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA
| | - Miranda J. Ridder
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Jeffrey L. Bose
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Kelly C. Rice
- Department of Microbiology & Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA
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15
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Wang N, Li P, Cheng Y, Song H, Xu C. Stem-loop structures control mRNA processing of the cellulosomal cip-cel operon in Ruminiclostridium cellulolyticum. BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS 2023; 16:106. [PMID: 37386549 DOI: 10.1186/s13068-023-02357-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/02/2022] [Accepted: 06/11/2023] [Indexed: 07/01/2023]
Abstract
BACKGROUND Anaerobic, mesophilic, and cellulolytic Ruminiclostridium cellulolyticum produces an efficient cellulolytic extracellular complex named cellulosome, which consist of a non-catalytic multi-functional integrating subunit, organizing the various catalytic subunits into the complex. Main components of cellulosome were encoded by the cip-cel operon in R. cellulolyticum, and their stoichiometry is controlled by the mechanism of selective RNA processing and stabilization, which allows to confer each processed RNA portion from the cip-cel mRNA on different fates due to their stability and resolve the potential contradiction between the equimolar stoichiometry of transcripts with a within a transcription unit and the non-equimolar stoichiometry of subunits. RESULTS In this work, RNA processing events were found to occur at six intergenic regions (IRs) harboring stem-loop structures in cip-cel operon. These stem-loops not only stabilize processed transcripts at their both ends, but also act as cleavage signals specifically recognized by endoribonucleases. We further demonstrated that cleavage sites were often located downstream or 3' end of their associated stem-loops that could be classified into two types, with distinct GC-rich stems being required for RNA cleavage. However, the cleavage site in IR4 was found to be located upstream of the stem-loop, as determined by the bottom AT-pair region of this stem-loop, together with its upstream structure. Thus, our findings reveal the structural requirements for processing of cip-cel transcripts, which can be potentially used to control the stoichiometry of gene expression in an operon. CONCLUSIONS Our findings reveal that stem-loop structures acting as RNA cleavage signals not only can be recognized by endoribonucleases and determine the location of cleavage sites but also determine the stoichiometry of their flanking processed transcripts by controlling stability in cip-cel operon. These features represent a complexed regulation of cellulosome in the post-transcriptional level, which can be exploited for designing synthetic elements to control gene expression.
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Affiliation(s)
- Na Wang
- Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, China-Australia Joint Laboratory for Animal Health Big Data Analytics, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, College of Animal Science and Technology & College of Veterinary Medicine, Zhejiang A&F University, Hangzhou, 311300, Zhejiang, China
- Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, 030006, Shanxi, China
| | - Ping Li
- Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, 030006, Shanxi, China
| | - Ying Cheng
- Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, 030006, Shanxi, China
| | - Houhui Song
- Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, China-Australia Joint Laboratory for Animal Health Big Data Analytics, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, College of Animal Science and Technology & College of Veterinary Medicine, Zhejiang A&F University, Hangzhou, 311300, Zhejiang, China.
| | - Chenggang Xu
- Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, China-Australia Joint Laboratory for Animal Health Big Data Analytics, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, College of Animal Science and Technology & College of Veterinary Medicine, Zhejiang A&F University, Hangzhou, 311300, Zhejiang, China.
- Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, 030006, Shanxi, China.
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16
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Korobeinikova A, Laalami S, Berthy C, Putzer H. RNase Y Autoregulates Its Synthesis in Bacillus subtilis. Microorganisms 2023; 11:1374. [PMID: 37374876 DOI: 10.3390/microorganisms11061374] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2023] [Revised: 05/18/2023] [Accepted: 05/22/2023] [Indexed: 06/29/2023] Open
Abstract
The instability of messenger RNA is crucial to the control of gene expression. In Bacillus subtilis, RNase Y is the major decay-initiating endoribonuclease. Here, we show how this key enzyme regulates its own synthesis by modulating the longevity of its mRNA. Autoregulation is achieved through cleavages in two regions of the rny (RNase Y) transcript: (i) within the first ~100 nucleotides of the open reading frame, immediately inactivating the mRNA for further rounds of translation; (ii) cleavages in the rny 5' UTR, primarily within the 5'-terminal 50 nucleotides, creating entry sites for the 5' exonuclease J1 whose progression is blocked around position -15 of the rny mRNA, potentially by initiating ribosomes. This links the functional inactivation of the transcript by RNase J1 to translation efficiency, depending on the ribosome occupancy at the translation initiation site. By these mechanisms, RNase Y can initiate degradation of its own mRNA when the enzyme is not occupied with degradation of other RNAs and thus prevent its overexpression beyond the needs of RNA metabolism.
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Affiliation(s)
- Anna Korobeinikova
- Expression Génétique Microbienne, Institut de Biologie Physico-Chimique, CNRS, Université Paris Cité, 75005 Paris, France
| | - Soumaya Laalami
- Expression Génétique Microbienne, Institut de Biologie Physico-Chimique, CNRS, Université Paris Cité, 75005 Paris, France
| | - Clément Berthy
- Expression Génétique Microbienne, Institut de Biologie Physico-Chimique, CNRS, Université Paris Cité, 75005 Paris, France
- Inovarion, 75005 Paris, France
| | - Harald Putzer
- Expression Génétique Microbienne, Institut de Biologie Physico-Chimique, CNRS, Université Paris Cité, 75005 Paris, France
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17
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Huch S, Nersisyan L, Ropat M, Barrett D, Wu M, Wang J, Valeriano VD, Vardazaryan N, Huerta-Cepas J, Wei W, Du J, Steinmetz LM, Engstrand L, Pelechano V. Atlas of mRNA translation and decay for bacteria. Nat Microbiol 2023:10.1038/s41564-023-01393-z. [PMID: 37217719 DOI: 10.1038/s41564-023-01393-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2022] [Accepted: 04/19/2023] [Indexed: 05/24/2023]
Abstract
Regulation of messenger RNA stability is pivotal for programmed gene expression in bacteria and is achieved by a myriad of molecular mechanisms. By bulk sequencing of 5' monophosphorylated mRNA decay intermediates (5'P), we show that cotranslational mRNA degradation is conserved among both Gram-positive and -negative bacteria. We demonstrate that, in species with 5'-3' exonucleases, the exoribonuclease RNase J tracks the trailing ribosome to produce an in vivo single-nucleotide toeprint of the 5' position of the ribosome. In other species lacking 5'-3' exonucleases, ribosome positioning alters endonucleolytic cleavage sites. Using our metadegradome (5'P degradome) sequencing approach, we characterize 5'P mRNA decay intermediates in 96 species including Bacillus subtilis, Escherichia coli, Synechocystis spp. and Prevotella copri and identify codon- and gene-level ribosome stalling responses to stress and drug treatment. We also apply 5'P sequencing to complex clinical and environmental microbiomes and demonstrate that metadegradome sequencing provides fast, species-specific posttranscriptional characterization of responses to drug or environmental perturbations. Finally we produce a degradome atlas for 96 species to enable analysis of mechanisms of RNA degradation in bacteria. Our work paves the way for the application of metadegradome sequencing to investigation of posttranscriptional regulation in unculturable species and complex microbial communities.
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Affiliation(s)
- Susanne Huch
- SciLifeLab, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden
| | - Lilit Nersisyan
- SciLifeLab, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden
- Armenian Bioinformatics Institute, Yerevan, Armenia
- Institute of Molecular Biology, National Academy of Sciences of Armenia, Yerevan, Armenia
| | - Maria Ropat
- SciLifeLab, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden
| | - Donal Barrett
- SciLifeLab, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden
| | - Mengjun Wu
- SciLifeLab, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden
| | - Jing Wang
- Department of Microbiology, Tumor and Cell Biology, Centre for Translational Microbiome Research, Karolinska Institutet, Stockholm, Sweden
| | - Valerie D Valeriano
- Department of Microbiology, Tumor and Cell Biology, Centre for Translational Microbiome Research, Karolinska Institutet, Stockholm, Sweden
| | - Nelli Vardazaryan
- Armenian Bioinformatics Institute, Yerevan, Armenia
- Institute of Molecular Biology, National Academy of Sciences of Armenia, Yerevan, Armenia
| | - Jaime Huerta-Cepas
- Centro de Biotecnologia y Genomica de Plantas, Universidad Politécnica de Madrid - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Campus de Montegancedo-UPM, Madrid, Spain
| | - Wu Wei
- Bio-Med Big Data Center, CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China
| | - Juan Du
- Department of Microbiology, Tumor and Cell Biology, Centre for Translational Microbiome Research, Karolinska Institutet, Stockholm, Sweden
| | - Lars M Steinmetz
- Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA
- Department of Genetics, School of Medicine, Stanford University, Stanford, CA, USA
- European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany
| | - Lars Engstrand
- Department of Microbiology, Tumor and Cell Biology, Centre for Translational Microbiome Research, Karolinska Institutet, Stockholm, Sweden
| | - Vicent Pelechano
- SciLifeLab, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden.
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18
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Song E, Hwang S, Munasingha PR, Seo YS, Kang J, Kang C, Hohng S. Transcriptional pause extension benefits the stand-by rather than catch-up Rho-dependent termination. Nucleic Acids Res 2023; 51:2778-2789. [PMID: 36762473 PMCID: PMC10085680 DOI: 10.1093/nar/gkad051] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2022] [Revised: 12/30/2022] [Accepted: 01/19/2023] [Indexed: 02/11/2023] Open
Abstract
Transcriptional pause is essential for all types of termination. In this single-molecule study on bacterial Rho factor-dependent terminators, we confirm that the three Rho-dependent termination routes operate compatibly together in a single terminator, and discover that their termination efficiencies depend on the terminational pauses in unexpected ways. Evidently, the most abundant route is that Rho binds nascent RNA first and catches up with paused RNA polymerase (RNAP) and this catch-up Rho mediates simultaneous releases of transcript RNA and template DNA from RNAP. The fastest route is that the catch-up Rho effects RNA-only release and leads to 1D recycling of RNAP on DNA. The slowest route is that the RNAP-prebound stand-by Rho facilitates only the simultaneous rather than sequential releases. Among the three routes, only the stand-by Rho's termination efficiency positively correlates with pause duration, contrary to a long-standing speculation, invariably in the absence or presence of NusA/NusG factors, competitor RNAs or a crowding agent. Accordingly, the essential terminational pause does not need to be long for the catch-up Rho's terminations, and long pauses benefit only the stand-by Rho's terminations. Furthermore, the Rho-dependent termination of mgtA and ribB riboswitches is controlled mainly by modulation of the stand-by rather than catch-up termination.
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Affiliation(s)
- Eunho Song
- Department of Physics and Astronomy, and Institute of Applied Physics, Seoul National University, Seoul 08826, Republic of Korea
| | - Seungha Hwang
- Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea
| | - Palinda Ruvan Munasingha
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea
| | - Yeon-Soo Seo
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea
| | - Jin Young Kang
- Correspondence may also be addressed to Jin Young Kang. Tel: +82 42 350 2831;
| | - Changwon Kang
- Correspondence may also be addressed to Changwon Kang. Tel: +82 42 350 2610;
| | - Sungchul Hohng
- To whom correspondence should be addressed. Tel: +82 2 880 6593;
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19
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Weiss CA, Myers TM, Wu CH, Jenkins C, Sondermann H, Lee V, Winkler WC. NrnA is a 5'-3' exonuclease that processes short RNA substrates in vivo and in vitro. Nucleic Acids Res 2022; 50:12369-12388. [PMID: 36478094 PMCID: PMC9757072 DOI: 10.1093/nar/gkac1091] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Revised: 10/25/2022] [Accepted: 11/30/2022] [Indexed: 12/12/2022] Open
Abstract
Bacterial RNases process RNAs until only short oligomers (2-5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2-4 nucleotides in length, all of which were processed from their 5' terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.
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Affiliation(s)
| | | | - Chih Hao Wu
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA
| | - Conor Jenkins
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
| | - Holger Sondermann
- CSSB – Centre for Structural Systems Biology, Deutsches Elektronen-Synchrotron (DESY), 22607 Hamburg, Germany,Christian-Albrechts-Universität, 24118 Kiel, Germany
| | - Vincent T Lee
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA
| | - Wade C Winkler
- To whom correspondence should be addressed. Tel: +1 301 405 7780;
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20
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Structural Insights into the Dimeric Form of Bacillus subtilis RNase Y Using NMR and AlphaFold. Biomolecules 2022; 12:biom12121798. [PMID: 36551226 PMCID: PMC9775385 DOI: 10.3390/biom12121798] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2022] [Revised: 11/23/2022] [Accepted: 11/28/2022] [Indexed: 12/04/2022] Open
Abstract
RNase Y is a crucial component of genetic translation, acting as the key enzyme initiating mRNA decay in many Gram-positive bacteria. The N-terminal domain of Bacillus subtilis RNase Y (Nter-BsRNaseY) is thought to interact with various protein partners within a degradosome complex. Bioinformatics and biophysical analysis have previously shown that Nter-BsRNaseY, which is in equilibrium between a monomeric and a dimeric form, displays an elongated fold with a high content of α-helices. Using multidimensional heteronuclear NMR and AlphaFold models, here, we show that the Nter-BsRNaseY dimer is constituted of a long N-terminal parallel coiled-coil structure, linked by a turn to a C-terminal region composed of helices that display either a straight or bent conformation. The structural organization of the N-terminal domain is maintained within the AlphaFold model of the full-length RNase Y, with the turn allowing flexibility between the N- and C-terminal domains. The catalytic domain is globular, with two helices linking the KH and HD modules, followed by the C-terminal region. This latter region, with no function assigned up to now, is most likely involved in the dimerization of B. subtilis RNase Y together with the N-terminal coiled-coil structure.
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21
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Carpousis AJ, Campo N, Hadjeras L, Hamouche L. Compartmentalization of RNA Degradosomes in Bacteria Controls Accessibility to Substrates and Ensures Concerted Degradation of mRNA to Nucleotides. Annu Rev Microbiol 2022; 76:533-552. [PMID: 35671533 DOI: 10.1146/annurev-micro-041020-113308] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
RNA degradosomes are multienzyme complexes composed of ribonucleases, RNA helicases, and metabolic enzymes. RNase E-based degradosomes are widespread in Proteobacteria. The Escherichia coli RNA degradosome is sequestered from transcription in the nucleoid and translation in the cytoplasm by localization to the inner cytoplasmic membrane, where it forms short-lived clusters that are proposed to be sites of mRNA degradation. In Caulobacter crescentus, RNA degradosomes localize to ribonucleoprotein condensates in the interior of the cell [bacterial ribonucleoprotein-bodies (BR-bodies)], which have been proposed to drive the concerted degradation of mRNA to nucleotides. The turnover of mRNA in growing cells is important for maintaining pools of nucleotides for transcription and DNA replication. Membrane attachment of the E. coli RNA degradosome is necessary to avoid wasteful degradation of intermediates in ribosome assembly. Sequestering RNA degradosomes to C. crescentus BR-bodies, which exclude structured RNA, could have a similar role in protecting intermediates in ribosome assembly from degradation. Expected final online publication date for the Annual Review of Microbiology, Volume 76 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
- Agamemnon J Carpousis
- LMGM, Université de Toulouse, CNRS, UPS, CBI, Toulouse, France; , , .,TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France
| | - Nathalie Campo
- LMGM, Université de Toulouse, CNRS, UPS, CBI, Toulouse, France; , ,
| | - Lydia Hadjeras
- LMGM, Université de Toulouse, CNRS, UPS, CBI, Toulouse, France; , , .,Current affiliation: IMIB, University of Würzburg, Würzburg, Germany;
| | - Lina Hamouche
- LMGM, Université de Toulouse, CNRS, UPS, CBI, Toulouse, France; , ,
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22
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Chhabra S, Mandell ZF, Liu B, Babitzke P, Bechhofer DH. Analysis of mRNA Decay Intermediates in Bacillus subtilis 3' Exoribonuclease and RNA Helicase Mutant Strains. mBio 2022; 13:e0040022. [PMID: 35311531 PMCID: PMC9040804 DOI: 10.1128/mbio.00400-22] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2022] [Accepted: 02/28/2022] [Indexed: 12/22/2022] Open
Abstract
The Bacillus subtilis genome encodes four 3' exoribonucleases: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Previous work showed that PNPase, encoded by the pnpA gene, is the major 3' exonuclease involved in mRNA turnover; in a pnpA deletion strain, numerous mRNA decay intermediates accumulate. Whether B. subtilis mRNA decay occurs in the context of a degradosome complex is controversial. In this study, global mapping of mRNA decay intermediate 3' ends within coding sequences was performed in strains that were either deleted for or had an inactivating point mutation in the pnpA gene. The patterns of 3'-end accumulation in these strains were highly similar, which may have implications for the role of a degradosome in mRNA decay. A comparison with mapped 3' ends in a strain lacking CshA, the major RNA helicase, indicated that many mRNAs require both PNPase and CshA for efficient decay. Transcriptome sequencing (RNA-seq) analysis of strains lacking RNase R suggested that this enzyme did not play a major role in mRNA turnover in the wild-type strain. Strains were constructed that contained only one of the four known 3' exoribonucleases. When RNase R was the only 3' exonuclease present, it was able to degrade a model mRNA efficiently, showing processive decay even through a strong stem-loop structure that inhibits PNPase processivity. Strains containing only RNase PH or only YhaM were also insensitive to this RNA secondary structure, suggesting the existence of another, as-yet-unidentified, 3' exoribonuclease. IMPORTANCE The ability to rapidly change bacterial gene expression programs in response to environmental conditions is highly dependent on the efficient turnover of mRNA. While much is known about the regulation of gene expression at the transcriptional and translational levels, much less is known about the intermediate step of mRNA decay. Here, we mapped the 3' ends of mRNA decay intermediates in strains that were missing the major 3' exoribonuclease PNPase or the RNA helicase CshA. We also assessed the roles of three other B. subtilis 3' exonucleases in the mRNA decay process. The data confirm the primary role of PNPase in mRNA turnover and suggest the involvement of one or more unknown RNases.
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Affiliation(s)
- Shivani Chhabra
- Icahn School of Medicine at Mount Sinai, Department of Pharmacological Sciences, New York, New York, USA
| | - Zachary F. Mandell
- The Pennsylvania State University, Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, University Park, Pennsylvania, USA
| | - Bo Liu
- Icahn School of Medicine at Mount Sinai, Department of Pharmacological Sciences, New York, New York, USA
| | - Paul Babitzke
- The Pennsylvania State University, Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, University Park, Pennsylvania, USA
| | - David H. Bechhofer
- Icahn School of Medicine at Mount Sinai, Department of Pharmacological Sciences, New York, New York, USA
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23
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Abstract
RNase J exerts both 5'-3' exoribonuclease and endoribonuclease activities and plays a major role in ribonucleotide metabolism in various bacteria; however, its gene regulation is not well understood. In this study, we investigated the regulation of rnj expression in Corynebacterium glutamicum. rnj mRNA expression was increased in a strain with an rnj mutation. Deletion of the genes encoding RNase E/G also resulted in increased rnj mRNA levels, although the effect was smaller than that of the rnj mutation. rnj mRNA was more stable in the rnj mutant strain than in wild-type cells. These results indicate that RNase J regulates its own gene by degrading its mRNA. The growth of rnj and pnp mutant cells was impaired at cold temperatures. The expression of rnj mRNA was transiently induced by cold shock; however, this induction was not observed in the rnj mutant strain, suggesting that autoregulation by self-degradation is responsible for inducing of rnj expression under cold-shock conditions. IMPORTANCE Corynebacterium glutamicum harbors one RNase E/G-type enzyme and one RNase J-type enzyme which are major ribonucleases in various bacteria. However, little is known about these gene regulations. Here, we show that RNase J autoregulates its own gene expression and RNase E/G is also involved in the rnj mRNA degradation. Furthermore, we show that transient induction of the rnj mRNA in the cold-shock condition is dependent on RNase J autoregulation. This study sheds light on the regulatory mechanism of RNase J in C. glutamicum.
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24
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Hinrichs R, Pozhydaieva N, Höfer K, Graumann PL. Y-Complex Proteins Show RNA-Dependent Binding Events at the Cell Membrane and Distinct Single-Molecule Dynamics. Cells 2022; 11:cells11060933. [PMID: 35326384 PMCID: PMC8945944 DOI: 10.3390/cells11060933] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Revised: 03/03/2022] [Accepted: 03/04/2022] [Indexed: 02/07/2023] Open
Abstract
Bacteria are dependent on rapid alterations in gene expression. A prerequisite for rapid adaptations is efficient RNA turnover, with endonuclease RNase Y playing a crucial role in mRNA stability as well as in maturation. In Bacillus subtilis, RNase Y in turn interacts with the so-called “Y-complex” consisting of three proteins, which play important functions in sporulation, natural transformation and biofilm formation. It is thought that the Y-complex acts as an accessory factor in RNase Y regulation but might also have independent functions. Using single-molecule tracking, we show that all three Y-complex proteins exhibit three distinct mobilities, including movement through the cytosol and confined motion, predominantly at membrane-proximal sites but also within the cell center. A transcriptional arrest leads to a strong change in localization and dynamics of YmcA, YlbF and YaaT, supporting their involvement in global RNA degradation. However, Y-complex proteins show distinguishable protein dynamics, and the deletion of yaaT or ylbF shows a minor effect on the dynamics of YmcA. Cell fractionation reveals that YaaT displays a mixture of membrane association and presence in the cytosol, while YlbF and YmcA do not show direct membrane attachment. Taken together, our experiments reveal membrane-associated and membrane-independent activities of Y-complex proteins and a dynamic interplay between them with indirect membrane association of YmcA and YlbF via YaaT.
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Affiliation(s)
- Rebecca Hinrichs
- SYNMIKRO, Zentrum für Synthetische Mikrobiologie, Philipps Universität Marburg, Karl-von-Frisch-Str. 14, 35043 Marburg, Germany; (R.H.); (N.P.); (K.H.)
- Fachbereich Chemie, Philipps Universität Marburg, Hans-Meerwein-Straße 4, 35043 Marburg, Germany
| | - Nadiia Pozhydaieva
- SYNMIKRO, Zentrum für Synthetische Mikrobiologie, Philipps Universität Marburg, Karl-von-Frisch-Str. 14, 35043 Marburg, Germany; (R.H.); (N.P.); (K.H.)
- Max-Planck-Institut für Terrestrische Mikrobiologie, Karl-von-Frisch Straße 16, 35043 Marburg, Germany
| | - Katharina Höfer
- SYNMIKRO, Zentrum für Synthetische Mikrobiologie, Philipps Universität Marburg, Karl-von-Frisch-Str. 14, 35043 Marburg, Germany; (R.H.); (N.P.); (K.H.)
- Max-Planck-Institut für Terrestrische Mikrobiologie, Karl-von-Frisch Straße 16, 35043 Marburg, Germany
| | - Peter L. Graumann
- SYNMIKRO, Zentrum für Synthetische Mikrobiologie, Philipps Universität Marburg, Karl-von-Frisch-Str. 14, 35043 Marburg, Germany; (R.H.); (N.P.); (K.H.)
- Fachbereich Chemie, Philipps Universität Marburg, Hans-Meerwein-Straße 4, 35043 Marburg, Germany
- Correspondence: ; Tel.: +49-6421-282-2210
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25
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Olejniczak M, Jiang X, Basczok MM, Storz G. KH domain proteins: Another family of bacterial RNA matchmakers? Mol Microbiol 2022; 117:10-19. [PMID: 34748246 PMCID: PMC8766902 DOI: 10.1111/mmi.14842] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Revised: 11/03/2021] [Accepted: 11/03/2021] [Indexed: 01/03/2023]
Abstract
In many bacteria, the stabilities and functions of small regulatory RNAs (sRNAs) that act by base pairing with target RNAs most often are dependent on Hfq or ProQ/FinO-domain proteins, two classes of RNA chaperone proteins. However, while all bacteria appear to have sRNAs, many have neither Hfq nor ProQ/FinO-domain proteins raising the question of whether another factor might act as an sRNA chaperone in these organisms. Several recent studies have reported that KH domain proteins, such as KhpA and KhpB, bind sRNAs. Here we describe what is known about the distribution, structures, RNA-binding properties, and physiologic roles of KhpA and KhpB and discuss evidence for and against these proteins serving as sRNAs chaperones.
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Affiliation(s)
- Mikolaj Olejniczak
- Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Uniwersytetu Poznańskiego 6, 61-614 Poznań, Poland
| | - Xiaofang Jiang
- Intramural Research Program, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA
| | - Maciej M. Basczok
- Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Uniwersytetu Poznańskiego 6, 61-614 Poznań, Poland
| | - Gisela Storz
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institutes of Child Health and Human Development, Bethesda, MD 20892-4417, USA
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26
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Condon C, Pellegrini O, Gilet L, Durand S, Braun F. Walking from E. coli to B. subtilis, one ribonuclease at a time. C R Biol 2021; 344:357-371. [DOI: 10.5802/crbiol.70] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2021] [Accepted: 11/22/2021] [Indexed: 11/24/2022]
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27
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Oviedo-Bocanegra LM, Hinrichs R, Rotter DAO, Dersch S, Graumann PL. Single molecule/particle tracking analysis program SMTracker 2.0 reveals different dynamics of proteins within the RNA degradosome complex in Bacillus subtilis. Nucleic Acids Res 2021; 49:e112. [PMID: 34417617 PMCID: PMC8565344 DOI: 10.1093/nar/gkab696] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2021] [Accepted: 07/29/2021] [Indexed: 01/06/2023] Open
Abstract
Single-molecule (particle) tracking is a powerful method to study dynamic processes in cells at highest possible spatial and temporal resolution. We have developed SMTracker, a graphical user interface for automatic quantifying, visualizing and managing of data. Version 2.0 determines distributions of positional displacements in x- and y-direction using multi-state diffusion models, discriminates between Brownian, sub- or superdiffusive behaviour, and locates slow or fast diffusing populations in a standardized cell. Using SMTracker, we show that the Bacillus subtilis RNA degradosome consists of a highly dynamic complex of RNase Y and binding partners. We found similar changes in molecule dynamics for RNase Y, CshA, PNPase and enolase, but not for phosphofructokinase, RNase J1 and J2, to inhibition of transcription. However, the absence of PfkA or of RNase J2 affected molecule dynamics of RNase Y-mVenus, indicating that these two proteins are indeed part of the degradosome. Molecule counting suggests that RNase Y is present as a dimer in cells, at an average copy number of about 500, of which 46% are present in a slow-diffusive state and thus likely engaged within degradosomes. Thus, RNase Y, CshA, PNPase and enolase likely play central roles, and RNase J1, J2 and PfkA more peripheral roles, in degradosome architecture.
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Affiliation(s)
- Luis M Oviedo-Bocanegra
- Centre for Synthetic Microbiology (SYNMIKRO) and Fachbereich Chemie, Philipps-Universität Marburg, 35032 Marburg, Germany
| | - Rebecca Hinrichs
- Centre for Synthetic Microbiology (SYNMIKRO) and Fachbereich Chemie, Philipps-Universität Marburg, 35032 Marburg, Germany
| | - Daniel Andreas Orlando Rotter
- Centre for Synthetic Microbiology (SYNMIKRO) and Fachbereich Chemie, Philipps-Universität Marburg, 35032 Marburg, Germany
| | - Simon Dersch
- Centre for Synthetic Microbiology (SYNMIKRO) and Fachbereich Chemie, Philipps-Universität Marburg, 35032 Marburg, Germany
| | - Peter L Graumann
- Centre for Synthetic Microbiology (SYNMIKRO) and Fachbereich Chemie, Philipps-Universität Marburg, 35032 Marburg, Germany
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28
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Abstract
RNases perform indispensable functions in regulating gene expression in many bacterial pathogens by processing and/or degrading RNAs. Despite the pivotal role of RNases in regulating bacterial virulence factors, the functions of RNases have not yet been studied in the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus). Here, we sought to determine the impact of two conserved RNases, the endoribonuclease RNase Y and exoribonuclease polynucleotide phosphorylase (PNPase), on the physiology and virulence of S. pneumoniae serotype 2 strain D39. We report that RNase Y and PNPase are essential for pneumococcal pathogenesis, as both deletion mutants showed strong attenuation of virulence in murine models of invasive pneumonia. Genome-wide transcriptomic analysis revealed that the abundances of nearly 200 mRNA transcripts were significantly increased, whereas those of several pneumococcal small regulatory RNAs (sRNAs), including the Ccn (CiaR-controlled noncoding RNA) sRNAs, were altered in the Δrny mutant relative to the wild-type strain. Additionally, lack of RNase Y resulted in pleiotropic phenotypes that included defects in pneumococcal cell morphology and growth in vitro. In contrast, Δpnp mutants showed no growth defect in vitro but differentially expressed a total of 40 transcripts, including the tryptophan biosynthesis operon genes and numerous 5' cis-acting regulatory RNAs, a majority of which were previously shown to impact pneumococcal disease progression in mice using the serotype 4 strain TIGR4. Together, our data suggest that RNase Y exerts a global impact on pneumococcal physiology, while PNPase mediates virulence phenotypes, likely through sRNA regulation. IMPORTANCE Streptococcus pneumoniae is a notorious human pathogen that adapts to conditions in distinct host tissues and responds to host cell interactions by adjusting gene expression. RNases are key players that modulate gene expression by mediating the turnover of regulatory and protein-coding transcripts. Here, we characterized two highly conserved RNases, RNase Y and PNPase, and evaluated their impact on the S. pneumoniae transcriptome for the first time. We show that PNPase influences the levels of a narrow set of mRNAs but a large number of regulatory RNAs primarily implicated in virulence control, whereas RNase Y has a more sweeping effect on gene expression, altering levels of transcripts involved in diverse cellular processes, including cell division, metabolism, stress response, and virulence. This study further reveals that RNase Y regulates expression of genes governing competence by mediating the turnover of CiaR-controlled noncoding (Ccn) sRNAs.
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29
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Ariza-Mateos A, Nuthanakanti A, Serganov A. Riboswitch Mechanisms: New Tricks for an Old Dog. BIOCHEMISTRY (MOSCOW) 2021; 86:962-975. [PMID: 34488573 DOI: 10.1134/s0006297921080071] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Discovered almost twenty years ago, riboswitches turned out to be one of the most common regulatory systems in bacteria, with representatives found in eukaryotes and archaea. Unlike many other regulatory elements, riboswitches are entirely composed of RNA and capable of modulating expression of genes by direct binding of small cellular molecules. While bacterial riboswitches had been initially thought to control production of enzymes and transporters associated with small organic molecules via feedback regulatory circuits, later findings identified riboswitches directing expression of a wide range of genes and responding to various classes of molecules, including ions, signaling molecules, and others. The 5'-untranslated mRNA regions host a vast majority of riboswitches, which modulate transcription or translation of downstream genes through conformational rearrangements in the ligand-sensing domains and adjacent expression-controlling platforms. Over years, the repertoire of regulatory mechanisms employed by riboswitches has greatly expanded; most recent studies have highlighted the importance of alternative mechanisms, such as RNA degradation, for the riboswitch-mediated genetic circuits. This review discusses the plethora of bacterial riboswitch mechanisms and illustrates how riboswitches utilize different features and approaches to elicit various regulatory responses.
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Affiliation(s)
- Ascensión Ariza-Mateos
- Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY 10016, USA
| | - Ashok Nuthanakanti
- Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY 10016, USA
| | - Alexander Serganov
- Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY 10016, USA.
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30
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Abstract
Bacterial protein synthesis rates have evolved to maintain preferred stoichiometries at striking precision, from the components of protein complexes to constituents of entire pathways. Setting relative protein production rates to be well within a factor of two requires concerted tuning of transcription, RNA turnover, and translation, allowing many potential regulatory strategies to achieve the preferred output. The last decade has seen a greatly expanded capacity for precise interrogation of each step of the central dogma genome-wide. Here, we summarize how these technologies have shaped the current understanding of diverse bacterial regulatory architectures underpinning stoichiometric protein synthesis. We focus on the emerging expanded view of bacterial operons, which encode diverse primary and secondary mRNA structures for tuning protein stoichiometry. Emphasis is placed on how quantitative tuning is achieved. We discuss the challenges and open questions in the application of quantitative, genome-wide methodologies to the problem of precise protein production. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
- James C Taggart
- Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; ,
| | - Jean-Benoît Lalanne
- Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; , .,Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.,Current affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA;
| | - Gene-Wei Li
- Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; ,
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31
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Benda M, Woelfel S, Faßhauer P, Gunka K, Klumpp S, Poehlein A, Kálalová D, Šanderová H, Daniel R, Krásný L, Stülke J. Quasi-essentiality of RNase Y in Bacillus subtilis is caused by its critical role in the control of mRNA homeostasis. Nucleic Acids Res 2021; 49:7088-7102. [PMID: 34157109 PMCID: PMC8266666 DOI: 10.1093/nar/gkab528] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 05/28/2021] [Accepted: 06/08/2021] [Indexed: 01/18/2023] Open
Abstract
RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase—α, β, β′. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the β or β’ subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed a severe decrease in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.
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Affiliation(s)
- Martin Benda
- Department of General Microbiology, GZMB, Georg-August-University Göttingen, Göttingen, Germany
| | - Simon Woelfel
- Department of General Microbiology, GZMB, Georg-August-University Göttingen, Göttingen, Germany
| | - Patrick Faßhauer
- Department of General Microbiology, GZMB, Georg-August-University Göttingen, Göttingen, Germany
| | - Katrin Gunka
- Department of General Microbiology, GZMB, Georg-August-University Göttingen, Göttingen, Germany
| | - Stefan Klumpp
- Institute for the Dynamics of Complex Systems, Georg-August-University Göttingen, Göttingen, Germany
| | - Anja Poehlein
- Department of Genomic and Applied Microbiology & Göttingen Genomics Laboratory, GZMB, Georg-August-University Göttingen, Göttingen, Germany
| | - Debora Kálalová
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic
| | - Hana Šanderová
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic
| | - Rolf Daniel
- Department of Genomic and Applied Microbiology & Göttingen Genomics Laboratory, GZMB, Georg-August-University Göttingen, Göttingen, Germany
| | - Libor Krásný
- Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic
| | - Jörg Stülke
- Department of General Microbiology, GZMB, Georg-August-University Göttingen, Göttingen, Germany
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32
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Laalami S, Cavaiuolo M, Roque S, Chagneau C, Putzer H. Escherichia coli RNase E can efficiently replace RNase Y in Bacillus subtilis. Nucleic Acids Res 2021; 49:4643-4654. [PMID: 33788929 PMCID: PMC8096251 DOI: 10.1093/nar/gkab216] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Revised: 03/11/2021] [Accepted: 03/17/2021] [Indexed: 02/06/2023] Open
Abstract
RNase Y and RNase E are disparate endoribonucleases that govern global mRNA turnover/processing in the two evolutionary distant bacteria Bacillus subtilis and Escherichia coli, respectively. The two enzymes share a similar in vitro cleavage specificity and subcellular localization. To evaluate the potential equivalence in biological function between the two enzymes in vivo we analyzed whether and to what extent RNase E is able to replace RNase Y in B. subtilis. Full-length RNase E almost completely restores wild type growth of the rny mutant. This is matched by a surprising reversal of transcript profiles both of individual genes and on a genome-wide scale. The single most important parameter to efficient complementation is the requirement for RNase E to localize to the inner membrane while truncation of the C-terminal sequences corresponding to the degradosome scaffold has only a minor effect. We also compared the in vitro cleavage activity for the major decay initiating ribonucleases Y, E and J and show that no conclusions can be drawn with respect to their activity in vivo. Our data confirm the notion that RNase Y and RNase E have evolved through convergent evolution towards a low specificity endonuclease activity universally important in bacteria.
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Affiliation(s)
- Soumaya Laalami
- CNRS, UMR8261, Institut de Biologie Physico-Chimique, Université de Paris, 75005 Paris, France
| | - Marina Cavaiuolo
- CNRS, UMR8261, Institut de Biologie Physico-Chimique, Université de Paris, 75005 Paris, France
| | - Sylvain Roque
- CNRS, UMR8261, Institut de Biologie Physico-Chimique, Université de Paris, 75005 Paris, France
| | - Carine Chagneau
- CNRS, UMR8261, Institut de Biologie Physico-Chimique, Université de Paris, 75005 Paris, France
| | - Harald Putzer
- CNRS, UMR8261, Institut de Biologie Physico-Chimique, Université de Paris, 75005 Paris, France
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33
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Abstract
Ribonucleases (RNases) are essential for almost every aspect of RNA metabolism. However, despite their important metabolic roles, RNases can also be destructive enzymes. As a consequence, cells must carefully regulate the amount, the activity, and the localization of RNases to avoid the inappropriate degradation of essential RNA molecules. In addition, bacterial cells often must adjust RNase levels as environmental situations demand, also requiring careful regulation of these critical enzymes. As the need for strict control of RNases has become more evident, multiple mechanisms for this regulation have been identified and studied, and these are described in this review. The major conclusion that emerges is that no common regulatory mechanism applies to all RNases, or even to a family of RNases; rather, a wide variety of processes have evolved that act on these enzymes, and in some cases, multiple regulatory mechanisms can even act on a single RNase. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
- Murray P Deutscher
- Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, Florida 33101, USA;
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Regulation of Glycine Cleavage and Detoxification by a Highly Conserved Glycine Riboswitch in Burkholderia spp. Curr Microbiol 2021; 78:2943-2955. [PMID: 34076709 DOI: 10.1007/s00284-021-02550-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2020] [Accepted: 05/21/2021] [Indexed: 10/21/2022]
Abstract
The glycine riboswitch is a known regulatory element that is unique in having two aptamers that are joined by a linker region. In this study, we investigated a glycine riboswitch located in the 5' untranslated region of a glycine cleavage system homolog (gcvTHP) in Burkholderia spp. Structure prediction using the sequence generated a model with a glycine binding pocket composed of base-triple interactions (G62-A64-A86 and G65-U84-C85) that are supported by A/G minor interactions (A17-C60-G88 and G16-C61-G87, respectively) and two ribose-zipper motifs (C11-G12 interacting with A248-A247 and C153-U154 interacting with A79-A78) which had not been previously reported. The capacity of the riboswitch to bind to glycine was experimentally validated by native gel assays and the crucial role of interactions that make up the glycine binding pocket were proven by mutations of A17U and G16C which resulted in conformational differences that may lead to dysfunction. Using glycine supplemented minimal media, we were able to prove that the expression of the gcvTHP genes found downstream of the riboswitch responded to the glycine concentrations introduced thus confirming the role of this highly conserved Burkholderia riboswitch and its associated genes as a putative glycine detoxification system in Burkholderia spp.
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35
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Moonlighting in Bacillus subtilis: The Small Proteins SR1P and SR7P Regulate the Moonlighting Activity of Glyceraldehyde 3-Phosphate Dehydrogenase A (GapA) and Enolase in RNA Degradation. Microorganisms 2021; 9:microorganisms9051046. [PMID: 34066298 PMCID: PMC8152036 DOI: 10.3390/microorganisms9051046] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2021] [Revised: 05/11/2021] [Accepted: 05/12/2021] [Indexed: 12/21/2022] Open
Abstract
Moonlighting proteins are proteins with more than one function. During the past 25 years, they have been found to be rather widespread in bacteria. In Bacillus subtilis, moonlighting has been disclosed to occur via DNA, protein or RNA binding or protein phosphorylation. In addition, two metabolic enzymes, enolase and phosphofructokinase, were localized in the degradosome-like network (DLN) where they were thought to be scaffolding components. The DLN comprises the major endoribonuclease RNase Y, 3'-5' exoribonuclease PnpA, endo/5'-3' exoribonucleases J1/J2 and helicase CshA. We have ascertained that the metabolic enzyme GapA is an additional component of the DLN. In addition, we identified two small proteins that bind scaffolding components of the degradosome: SR1P encoded by the dual-function sRNA SR1 binds GapA, promotes the GapA-RNase J1 interaction and increases the RNase J1 activity. SR7P encoded by the dual-function antisense RNA SR7 binds to enolase thereby enhancing the enzymatic activity of enolase bound RNase Y. We discuss the role of small proteins in modulating the activity of two moonlighting proteins.
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36
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Richards J, Belasco JG. Riboswitch control of bacterial RNA stability. Mol Microbiol 2021; 116:361-365. [PMID: 33797153 PMCID: PMC10367942 DOI: 10.1111/mmi.14723] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2021] [Revised: 03/25/2021] [Accepted: 03/29/2021] [Indexed: 11/30/2022]
Abstract
Although riboswitches have long been known to regulate translation initiation and transcription termination, a growing body of evidence indicates that they can also control bacterial RNA lifetimes by acting directly to hasten or impede RNA degradation. Ligand binding to the aptamer domain of a riboswitch can accelerate RNA decay by triggering a conformational change that exposes sites to endonucleolytic cleavage or by catalyzing the self-cleavage of a prefolded ribozyme. Alternatively, the conformational change induced by ligand binding can protect RNA from degradation by blocking access to an RNA terminus or internal region that would otherwise be susceptible to attack by an exonuclease or endonuclease. Such changes in RNA longevity often accompany a parallel effect of the same riboswitch on translation or transcription. Consequently, a single riboswitch aptamer may govern the function of multiple effector elements (expression platforms) that are co-resident within a transcript and act independently of one another.
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Affiliation(s)
- Jamie Richards
- Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA.,Department of Microbiology, New York University School of Medicine, New York, NY, USA
| | - Joel G Belasco
- Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA.,Department of Microbiology, New York University School of Medicine, New York, NY, USA
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37
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Wencker FDR, Marincola G, Schoenfelder SMK, Maaß S, Becher D, Ziebuhr W. Another layer of complexity in Staphylococcus aureus methionine biosynthesis control: unusual RNase III-driven T-box riboswitch cleavage determines met operon mRNA stability and decay. Nucleic Acids Res 2021; 49:2192-2212. [PMID: 33450025 PMCID: PMC7913692 DOI: 10.1093/nar/gkaa1277] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2020] [Revised: 12/18/2020] [Accepted: 01/08/2021] [Indexed: 11/12/2022] Open
Abstract
In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5′ untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5′-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3′-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements.
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Affiliation(s)
- Freya D R Wencker
- Institute of Molecular Infection Biology, University of Würzburg, Würzburg 97080, Germany
| | - Gabriella Marincola
- Institute of Molecular Infection Biology, University of Würzburg, Würzburg 97080, Germany
| | - Sonja M K Schoenfelder
- Institute of Molecular Infection Biology, University of Würzburg, Würzburg 97080, Germany
| | - Sandra Maaß
- Institute of Microbiology, University of Greifswald, Greifswald 17489, Germany
| | - Dörte Becher
- Institute of Microbiology, University of Greifswald, Greifswald 17489, Germany
| | - Wilma Ziebuhr
- Institute of Molecular Infection Biology, University of Würzburg, Würzburg 97080, Germany
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38
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Corsi ID, Dutta S, van Hoof A, Koehler TM. AtxA-Controlled Small RNAs of Bacillus anthracis Virulence Plasmid pXO1 Regulate Gene Expression in trans. Front Microbiol 2021; 11:610036. [PMID: 33519762 PMCID: PMC7843513 DOI: 10.3389/fmicb.2020.610036] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2020] [Accepted: 12/11/2020] [Indexed: 11/13/2022] Open
Abstract
Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been studied extensively in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. The sRNAs, named “XrrA and XrrB” (for pXO1-encoded regulatory RNA) are abundant and highly stable primary transcripts, whose expression is dependent upon AtxA, the master virulence regulator of B. anthracis. sRNA levels are highest during culture conditions that promote AtxA expression and activity, and sRNA levels are unaltered in Hfq RNA chaperone null-mutants. Comparison of the transcriptome of a virulent Ames-derived strain to the transcriptome of isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Most regulatory effects were associated with XrrA, although regulation of some transcripts suggests functional overlap between the XrrA and XrrB. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Finally, in a mouse model for systemic anthrax, the lungs and livers of animals infected with xrrA-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in B. anthracis pathogenesis.
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Affiliation(s)
- Ileana D Corsi
- Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, United States.,MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, The University of Texas, Houston, TX, United States
| | - Soumita Dutta
- Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, United States
| | - Ambro van Hoof
- Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, United States.,MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, The University of Texas, Houston, TX, United States
| | - Theresa M Koehler
- Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, United States.,MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, The University of Texas, Houston, TX, United States
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39
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Samuels DS, Lybecker MC, Yang XF, Ouyang Z, Bourret TJ, Boyle WK, Stevenson B, Drecktrah D, Caimano MJ. Gene Regulation and Transcriptomics. Curr Issues Mol Biol 2020; 42:223-266. [PMID: 33300497 DOI: 10.21775/cimb.042.223] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Borrelia (Borreliella) burgdorferi, along with closely related species, is the etiologic agent of Lyme disease. The spirochete subsists in an enzootic cycle that encompasses acquisition from a vertebrate host to a tick vector and transmission from a tick vector to a vertebrate host. To adapt to its environment and persist in each phase of its enzootic cycle, B. burgdorferi wields three systems to regulate the expression of genes: the RpoN-RpoS alternative sigma factor cascade, the Hk1/Rrp1 two-component system and its product c-di-GMP, and the stringent response mediated by RelBbu and DksA. These regulatory systems respond to enzootic phase-specific signals and are controlled or fine- tuned by transcription factors, including BosR and BadR, as well as small RNAs, including DsrABb and Bb6S RNA. In addition, several other DNA-binding and RNA-binding proteins have been identified, although their functions have not all been defined. Global changes in gene expression revealed by high-throughput transcriptomic studies have elucidated various regulons, albeit technical obstacles have mostly limited this experimental approach to cultivated spirochetes. Regardless, we know that the spirochete, which carries a relatively small genome, regulates the expression of a considerable number of genes required for the transitions between the tick vector and the vertebrate host as well as the adaptation to each.
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Affiliation(s)
- D Scott Samuels
- Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA
| | - Meghan C Lybecker
- Department of Biology, University of Colorado, Colorado Springs, CO 80918, USA
| | - X Frank Yang
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Zhiming Ouyang
- Department of Molecular Medicine, University of South Florida, Tampa, FL 33612, USA
| | - Travis J Bourret
- Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE, 68105 USA
| | - William K Boyle
- Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE, 68105 USA
| | - Brian Stevenson
- Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky School of Medicine, Lexington, KY 40536, USA
| | - Dan Drecktrah
- Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA
| | - Melissa J Caimano
- Departments of Medicine, Pediatrics, and Molecular Biology and Biophysics, UConn Health, Farmington, CT, USA
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40
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Raj R, Nadig S, Patel T, Gopal B. Structural and biochemical characteristics of two Staphylococcus epidermidis RNase J paralogs RNase J1 and RNase J2. J Biol Chem 2020; 295:16863-16876. [PMID: 32994223 PMCID: PMC7864078 DOI: 10.1074/jbc.ra120.014876] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2020] [Revised: 09/21/2020] [Indexed: 11/06/2022] Open
Abstract
RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.
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Affiliation(s)
- Rishi Raj
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
| | - Savitha Nadig
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
| | - Twinkal Patel
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
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41
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Zhang J, Xu X, Li X, Chen X, Zhou C, Liu Y, Li Y, Lu F. Reducing the cell lysis to enhance yield of acid-stable alpha amylase by deletion of multiple peptidoglycan hydrolase-related genes in Bacillus amyloliquefaciens. Int J Biol Macromol 2020; 167:777-786. [PMID: 33278447 DOI: 10.1016/j.ijbiomac.2020.11.193] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2020] [Revised: 11/26/2020] [Accepted: 11/29/2020] [Indexed: 11/30/2022]
Abstract
Bacillus amyloliquefaciens is a major industrial host for extracellular protein production, with great potential in the enzyme industry. However, the strain has accelerated the autolysis drawback in the process of secreting extracellular enzymes, which can significantly lower the density of cells and decrease the product yield. To identify target genes, we employed comparative transcriptome sequencing and KEGG analysis to indicate the increased expression of peptidoglycan hydrolase-regulated genes from the exponential phase to the apoptotic phase of growth; this was further confirmed by quantitative RT-PCR. By deleting lytD, lytE, and sigD genes, cell lysis was reduced and the production of acid-stable Bacillus licheniformis alpha-amylase was enhanced. After 36 h of culture, multiple deletion mutant BA ΔSDE had significantly more viable cells compared to the control strain BA Δupp, and flow cytometry analysis indicated that 48.43% and 64.03% of the cells were lysed in cultures of BA ΔSDE and BA Δupp, respectively. In a 2-L fed-batch fermenter, viable cell number of the triple deletion mutant BA ΔSDE increased by 2.79 Log/cfu/mL, and the activity of acid-stable alpha-amylase increased by 48.4%, compared to BA Δupp. Systematic multiple peptidoglycan hydrolases deletion relieved the autolysis and increased the production of industrial enzymes, and provided a useful strategy for guiding efforts to manipulate the genomes of other B. amyloliquefaciens used for chassis host.
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Affiliation(s)
- Jinfang Zhang
- Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
| | - Xiaojian Xu
- Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
| | - Xinyue Li
- Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
| | - Xuejia Chen
- Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
| | - Cuixia Zhou
- School of Biology and Brewing Engineering, Taishan University, Taian 271018, PR China
| | - Yihan Liu
- Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
| | - Yu Li
- Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
| | - Fuping Lu
- Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
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42
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Drogalis LK, Batey RT. Requirements for efficient ligand-gated co-transcriptional switching in designed variants of the B. subtilis pbuE adenine-responsive riboswitch in E. coli. PLoS One 2020; 15:e0243155. [PMID: 33259551 PMCID: PMC7707468 DOI: 10.1371/journal.pone.0243155] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2020] [Accepted: 11/16/2020] [Indexed: 11/18/2022] Open
Abstract
Riboswitches, generally located in the 5'-leader of bacterial mRNAs, direct expression via a small molecule-dependent structural switch that informs the transcriptional or translational machinery. While the structure and function of riboswitch effector-binding (aptamer) domains have been intensely studied, only recently have the requirements for efficient linkage between small molecule binding and the structural switch in the cellular and co-transcriptional context begun to be actively explored. To address this aspect of riboswitch function, we have performed a structure-guided mutagenic analysis of the B. subtilis pbuE adenine-responsive riboswitch, one of the simplest riboswitches that employs a strand displacement switching mechanism to regulate transcription. Using a cell-based fluorescent protein reporter assay to assess ligand-dependent regulatory activity in E. coli, these studies revealed previously unrecognized features of the riboswitch. Within the aptamer domain, local and long-range conformational dynamics influenced by sequences within helices have a significant effect upon efficient regulatory switching. Sequence features of the expression platform including the pre-aptamer leader sequence, a toehold helix and an RNA polymerase pause site all serve to promote strong ligand-dependent regulation. By optimizing these features, we were able to improve the performance of the B. subtilis pbuE riboswitch in E. coli from 5.6-fold induction of reporter gene expression by the wild type riboswitch to over 120-fold in the top performing designed variant. Together, these data point to sequence and structural features distributed throughout the riboswitch required to strike a balance between rates of ligand binding, transcription and secondary structural switching via a strand exchange mechanism and yield new insights into the design of artificial riboswitches.
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MESH Headings
- Adenine/metabolism
- Aptamers, Nucleotide/chemistry
- Aptamers, Nucleotide/genetics
- Aptamers, Nucleotide/metabolism
- Bacillus subtilis/genetics
- Bacillus subtilis/metabolism
- Escherichia coli K12/genetics
- Genes, Reporter
- Genetic Variation
- Ligands
- Models, Genetic
- Models, Molecular
- Mutagenesis
- Nucleic Acid Conformation
- RNA, Bacterial/chemistry
- RNA, Bacterial/genetics
- RNA, Bacterial/metabolism
- Riboswitch/genetics
- Transcription, Genetic
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Affiliation(s)
- Lea K. Drogalis
- Department of Biochemistry, University of Colorado, Boulder, Colorado, United States of America
| | - Robert T. Batey
- Department of Biochemistry, University of Colorado, Boulder, Colorado, United States of America
- * E-mail:
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43
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Abstract
Posttranscriptional regulation is a major level of gene expression control in any cell. In bacteria, multiprotein machines called RNA degradosomes are central for RNA processing and degradation, and some were reported to be compartmentalized inside these organelleless cells. The minimal RNA degradosome of the important gastric pathogen Helicobacter pylori is composed of the essential ribonuclease RNase J and RhpA, its sole DEAD box RNA helicase, and plays a major role in the regulation of mRNA decay and adaptation to gastric colonization. Here, the subcellular localization of the H. pylori RNA degradosome was investigated using cellular fractionation and both confocal and superresolution microscopy. We established that RNase J and RhpA are peripheral inner membrane proteins and that this association was mediated neither by ribosomes nor by RNA nor by the RNase Y membrane protein. In live H. pylori cells, we observed that fluorescent RNase J and RhpA protein fusions assemble into nonpolar foci. We identified factors that regulate the formation of these foci without affecting the degradosome membrane association. Flotillin, a bacterial membrane scaffolding protein, and free RNA promote focus formation in H. pylori Finally, RNase J-GFP (RNase J-green fluorescent protein) molecules and foci in cells were quantified by three-dimensional (3D) single-molecule fluorescence localization microscopy. The number and size of the RNase J foci were found to be scaled with growth phase and cell volume as previously reported for eukaryotic ribonucleoprotein granules. In conclusion, we propose that membrane compartmentalization and the regulated clustering of RNase J-based degradosome hubs represent important levels of control of their activity and specificity.IMPORTANCE Helicobacter pylori is a bacterial pathogen that chronically colonizes the stomach of half of the human population worldwide. Infection by H. pylori can lead to the development of gastric pathologies such as ulcers and adenocarcinoma, which causes up to 800,000 deaths in the world each year. Persistent colonization by H. pylori relies on regulation of the expression of adaptation-related genes. One major level of such control is posttranscriptional regulation, which, in H. pylori, largely relies on a multiprotein molecular machine, an RNA degradosome, that we previously discovered. In this study, we established that the two protein partners of this machine are associated with the membrane of H. pylori Using cutting-edge microscopy, we showed that these complexes assemble into hubs whose formation is regulated by free RNA and scaled with bacterial size and growth phase. Organelleless cellular compartmentalization of molecular machines into hubs emerges as an important regulatory level in bacteria.
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44
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Vargas-Blanco DA, Shell SS. Regulation of mRNA Stability During Bacterial Stress Responses. Front Microbiol 2020; 11:2111. [PMID: 33013770 PMCID: PMC7509114 DOI: 10.3389/fmicb.2020.02111] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2020] [Accepted: 08/11/2020] [Indexed: 12/12/2022] Open
Abstract
Bacteria have a remarkable ability to sense environmental changes, swiftly regulating their transcriptional and posttranscriptional machinery as a response. Under conditions that cause growth to slow or stop, bacteria typically stabilize their transcriptomes in what has been shown to be a conserved stress response. In recent years, diverse studies have elucidated many of the mechanisms underlying mRNA degradation, yet an understanding of the regulation of mRNA degradation under stress conditions remains elusive. In this review we discuss the diverse mechanisms that have been shown to affect mRNA stability in bacteria. While many of these mechanisms are transcript-specific, they provide insight into possible mechanisms of global mRNA stabilization. To that end, we have compiled information on how mRNA fate is affected by RNA secondary structures; interaction with ribosomes, RNA binding proteins, and small RNAs; RNA base modifications; the chemical nature of 5' ends; activity and concentration of RNases and other degradation proteins; mRNA and RNase localization; and the stringent response. We also provide an analysis of reported relationships between mRNA abundance and mRNA stability, and discuss the importance of stress-associated mRNA stabilization as a potential target for therapeutic development.
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Affiliation(s)
- Diego A Vargas-Blanco
- Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA, United States
| | - Scarlet S Shell
- Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA, United States.,Program in Bioinformatics and Computational Biology, Worcester Polytechnic Institute, Worcester, MA, United States
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45
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Abstract
Ro60 ribonucleoproteins (RNPs), composed of the ring-shaped Ro 60-kDa (Ro60) protein and noncoding RNAs called Y RNAs, are present in all three domains of life. Ro60 was first described as an autoantigen in patients with rheumatic disease, and Ro60 orthologs have been identified in 3% to 5% of bacterial genomes, spanning the majority of phyla. Their functions have been characterized primarily in Deinococcus radiodurans, the first sequenced bacterium with a recognizable ortholog. In D. radiodurans, the Ro60 ortholog enhances the ability of 3'-to-5' exoribonucleases to degrade structured RNA during several forms of environmental stress. Y RNAs are regulators that inhibit or allow the interactions of Ro60 with other proteins and RNAs. Studies of Ro60 RNPs in other bacteria hint at additional functions, since the most conserved Y RNA contains a domain that is a close tRNA mimic and Ro60 RNPs are often encoded adjacent to components of RNA repair systems.
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Affiliation(s)
- Soyeong Sim
- RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA; , , ,
| | - Kevin Hughes
- RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA; , , ,
- Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, USA
| | - Xinguo Chen
- RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA; , , ,
| | - Sandra L Wolin
- RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA; , , ,
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46
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Abstract
Here, we describe SR7, a dual-function antisense RNA encoded on the Bacillus subtilis chromosome. This RNA was earlier described as SigB-dependent regulatory RNA S1136 and reported to reduce the amount of the small ribosomal subunit under ethanol stress. We found that the 5ʹ portion of SR7 encodes a small protein composed of 39 amino acids which we designated SR7P. It is translated from a 185 nt SigB-dependent mRNA under five different stress conditions and a longer SigB-independent RNA constitutively. About three-fold higher amounts of SR7P were detected in B. subtilis cells exposed to salt, ethanol, acid or heat stress. Co-elution experiments with SR7PC-FLAG and Far-Western blotting demonstrated that SR7P interacts with the glycolytic enzyme enolase. Enolase is a scaffolding component of the B. subtilis degradosome where it interacts with RNase Y and phosphofructokinase PfkA. We found that SR7P increases the amount of RNase Y bound to enolase without affecting PfkA. RNA does not bridge the SR7P-enolase-RNase Y interaction. In vitro-degradation assays with the known RNase Y substrates yitJ and rpsO mRNA revealed enhanced enzymatic activity of enolase-bound RNase Y in the presence of SR7P. Northern blots showed a major effect of enolase and a minor effect of SR7P on the half-life of rpsO mRNA indicating a fine-tuning role of SR7P in RNA degradation.
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Affiliation(s)
- Inam Ul Haq
- Friedrich-Schiller-Universität Jena, Matthias-Schleiden-Institut , AG Bakteriengenetik, Jena, Germany
| | - Peter Müller
- Friedrich-Schiller-Universität Jena, Matthias-Schleiden-Institut , AG Bakteriengenetik, Jena, Germany
| | - Sabine Brantl
- Friedrich-Schiller-Universität Jena, Matthias-Schleiden-Institut , AG Bakteriengenetik, Jena, Germany
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47
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Oikonomopoulos S, Bayega A, Fahiminiya S, Djambazian H, Berube P, Ragoussis J. Methodologies for Transcript Profiling Using Long-Read Technologies. Front Genet 2020; 11:606. [PMID: 32733532 PMCID: PMC7358353 DOI: 10.3389/fgene.2020.00606] [Citation(s) in RCA: 53] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Accepted: 05/19/2020] [Indexed: 12/28/2022] Open
Abstract
RNA sequencing using next-generation sequencing technologies (NGS) is currently the standard approach for gene expression profiling, particularly for large-scale high-throughput studies. NGS technologies comprise high throughput, cost efficient short-read RNA-Seq, while emerging single molecule, long-read RNA-Seq technologies have enabled new approaches to study the transcriptome and its function. The emerging single molecule, long-read technologies are currently commercially available by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), while new methodologies based on short-read sequencing approaches are also being developed in order to provide long range single molecule level information-for example, the ones represented by the 10x Genomics linked read methodology. The shift toward long-read sequencing technologies for transcriptome characterization is based on current increases in throughput and decreases in cost, making these attractive for de novo transcriptome assembly, isoform expression quantification, and in-depth RNA species analysis. These types of analyses were challenging with standard short sequencing approaches, due to the complex nature of the transcriptome, which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes, as well as the high sequence similarity of highly abundant species of RNA, such as rRNAs. Here we aim to focus on single molecule level sequencing technologies and single-cell technologies that, combined with perturbation tools, allow the analysis of complete RNA species, whether short or long, at high resolution. In parallel, these tools have opened new ways in understanding gene functions at the tissue, network, and pathway levels, as well as their detailed functional characterization. Analysis of the epi-transcriptome, including RNA methylation and modification and the effects of such modifications on biological systems is now enabled through direct RNA sequencing instead of classical indirect approaches. However, many difficulties and challenges remain, such as methodologies to generate full-length RNA or cDNA libraries from all different species of RNAs, not only poly-A containing transcripts, and the identification of allele-specific transcripts due to current error rates of single molecule technologies, while the bioinformatics analysis on long-read data for accurate identification of 5' and 3' UTRs is still in development.
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Affiliation(s)
- Spyros Oikonomopoulos
- McGill Genome Centre, Department of Human Genetics, McGill University, Montréal, QC, Canada
| | - Anthony Bayega
- McGill Genome Centre, Department of Human Genetics, McGill University, Montréal, QC, Canada
| | - Somayyeh Fahiminiya
- McGill Genome Centre, Department of Human Genetics, McGill University, Montréal, QC, Canada
| | - Haig Djambazian
- McGill Genome Centre, Department of Human Genetics, McGill University, Montréal, QC, Canada
| | - Pierre Berube
- McGill Genome Centre, Department of Human Genetics, McGill University, Montréal, QC, Canada
| | - Jiannis Ragoussis
- McGill Genome Centre, Department of Human Genetics, McGill University, Montréal, QC, Canada
- Department of Bioengineering, McGill University, Montréal, QC, Canada
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48
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Li J, Hou Y, Gu X, Yue L, Guo L, Li D, Dong X. A newly identified duplex RNA unwinding activity of archaeal RNase J depends on processive exoribonucleolysis coupled steric occlusion by its structural archaeal loops. RNA Biol 2020; 17:1480-1491. [PMID: 32552320 DOI: 10.1080/15476286.2020.1777379] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022] Open
Abstract
RNase J is a prokaryotic 5'-3' exo/endoribonuclease that functions in mRNA decay and rRNA maturation. Here, we report a novel duplex unwinding activity of mpy-RNase J, an archaeal RNase J from Methanolobus psychrophilus, which enables it to degrade duplex RNAs with hairpins up to 40 bp when linking a 5' single-stranded overhangs of ≥ 7 nt, corresponding to the RNA channel length. A 6-nt RNA-mpy-RNase J-S247A structure reveals the RNA-interacting residues and a steric barrier at the RNA channel entrance comprising two archaeal loops and two helices. Mutagenesis of the residues key to either exoribonucleolysis or RNA translocation diminished the duplex unwinding activity. Substitution of the residues in the steric barrier yielded stalled degradation intermediates at the duplex RNA regions. Thus, an exoribonucleolysis-driven and steric occlusion-based duplex unwinding mechanism was identified. The duplex unwinding activity confers mpy-RNase J the capability of degrading highly structured RNAs, including the bacterial REP RNA, and archaeal mRNAs, rRNAs, tRNAs, SRPs, RNase P and CD-box RNAs, providing an indicative of the potential key roles of mpy-RNase J in pleiotropic RNA metabolisms. Hydrolysis-coupled duplex unwinding activity was also detected in a bacterial RNase J, which may use a shared but slightly different unwinding mechanism from archaeal RNase Js, indicating that duplex unwinding is a common property of the prokaryotic RNase Js.
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Affiliation(s)
- Jie Li
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing, PR China.,Colleges of Life Sciences, University of Chinese Academy of Sciences , Beijing, China
| | - Yanjie Hou
- Institute of Biophysics, Chinese Academy of Sciences , Beijing, PR China
| | - Xien Gu
- School of Basic Medical Sciences, Hubei University of Medicine , Shiyan, China
| | - Lei Yue
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing, PR China.,Colleges of Life Sciences, University of Chinese Academy of Sciences , Beijing, China
| | - Lu Guo
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing, PR China
| | - Defeng Li
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing, PR China.,Colleges of Life Sciences, University of Chinese Academy of Sciences , Beijing, China
| | - Xiuzhu Dong
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing, PR China.,Colleges of Life Sciences, University of Chinese Academy of Sciences , Beijing, China
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49
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Cavaiuolo M, Chagneau C, Laalami S, Putzer H. Impact of RNase E and RNase J on Global mRNA Metabolism in the Cyanobacterium Synechocystis PCC6803. Front Microbiol 2020; 11:1055. [PMID: 32582060 PMCID: PMC7283877 DOI: 10.3389/fmicb.2020.01055] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2020] [Accepted: 04/29/2020] [Indexed: 01/18/2023] Open
Abstract
mRNA levels result from an equilibrium between transcription and degradation. Ribonucleases (RNases) facilitate the turnover of mRNA, which is an important way of controlling gene expression, allowing the cells to adjust transcript levels to a changing environment. In contrast to the heterotrophic model bacteria Escherichia coli and Bacillus subtilis, RNA decay has not been studied in detail in cyanobacteria. Synechocystis sp. PCC6803 encodes orthologs of both E. coli and B. subtilis RNases, including RNase E and RNase J, respectively. We show that in vitro Sy RNases E and J have an endonucleolytic cleavage specificity that is very similar between them and also compared to orthologous enzymes from E. coli, B. subtilis, and Chlamydomonas. Moreover, Sy RNase J displays a robust 5′-exoribonuclease activity similar to B. subtilis RNase J1, but unlike the evolutionarily related RNase J in chloroplasts. Both nucleases are essential and gene deletions could not be fully segregated in Synechocystis. We generated partially disrupted strains of Sy RNase E and J that were stable enough to allow for their growth and characterization. A transcriptome analysis of these strains partially depleted for RNases E and J, respectively, allowed to observe effects on specific transcripts. RNase E altered the expression of a larger number of chromosomal genes and antisense RNAs compared to RNase J, which rather affects endogenous plasmid encoded transcripts. Our results provide the first description of the main transcriptomic changes induced by the partial depletion of two essential ribonucleases in cyanobacteria.
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Affiliation(s)
- Marina Cavaiuolo
- UMR 8261, CNRS, Institut de Biologie Physico-Chimique, Université de Paris, Paris, France
| | - Carine Chagneau
- UMR 8261, CNRS, Institut de Biologie Physico-Chimique, Université de Paris, Paris, France
| | - Soumaya Laalami
- UMR 8261, CNRS, Institut de Biologie Physico-Chimique, Université de Paris, Paris, France
| | - Harald Putzer
- UMR 8261, CNRS, Institut de Biologie Physico-Chimique, Université de Paris, Paris, France
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50
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Rational engineering of transcriptional riboswitches leads to enhanced metabolite levels in Bacillus subtilis. Metab Eng 2020; 61:58-68. [PMID: 32413407 DOI: 10.1016/j.ymben.2020.05.002] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2020] [Revised: 04/17/2020] [Accepted: 05/02/2020] [Indexed: 12/26/2022]
Abstract
Many metabolic pathways in bacteria are regulated by metabolite sensing riboswitches that exert their control at the level of transcription employing a termination-antitermination mechanism. These riboswitches represent engineering targets to modulate expression of genes and operons relevant for the biotechnological production of commercially relevant compounds. We show that removal of the transcriptional riboswitches that control purine biosynthesis and riboflavin biosynthesis in Bacillus subtilis leads to auxotrophic strains. As an alternative, we report a rational approach for engineering transcriptional riboswitches independently from the availability of structural data. This approach consists in the identification and deletion of a key nucleotide sequence exclusively involved in transcription termination without affecting formation of other secondary and tertiary structures, which can be involved in other functions. To demonstrate the efficacy of our approach, we tested it with regard to deregulation of the purine and the riboflavin biosynthetic pathways in B. subtilis. Following validation of the engineered transcriptional riboswitches using specialized reporter strains, our approach was implemented into a B. subtilis wild-type strain employing CRISPR-Cas9 genome editing. The resulting purine and riboflavin production strains were characterized at the level of gene expression, metabolite synthesis and growth, and a substantial enhancement was measured at each level. Moreover, applying our approach to deregulate the purine pathway of an industrial riboflavin overproducing strain with impaired growth led to an increase in biomass by 53%, which resulted in an enhanced total production of riboflavin in the culture.
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